Many genes, both in prokaryotes and eukaryotes, are regulated post- transcriptionally. The long-term objectives of this research are to understand the roles of RNA conformation, and ligand-induced RNA conformational switches in modulating gene expression. The studies in this proposal are designed to increase understanding of the mechanisms of translational repression of the E. coli alpha operon by the ribosomal S4 protein and antisense oligonucleotides. Repression of translational initiation may occur when a ligand prevents initial association of the ribosomal 30s subunit with the messenger RNA or a step subsequent to initial binding. The S4 protein and antisense oligonucleotide repressors bind to different mRNA sequences in the alpha operon, and are expected to have different mechanisms of translational regulation. The repressor molecules may stabilize different secondary and tertiary structures within the mRNA which need to be unfolded for efficient translation to occur. The mechanisms of translational repression by these ligands will be determined by thermodynamic and kinetic analyses of in vitro rates of protein synthesis as functions of ribosome and repressor concentration. Comparison of the results for different repressor molecules will provide insight into the functions of mRNA structure and RNA conformational switches. Additionally, this study will provide a quantitative understanding of a novel translational repression mechanism.